Human respiratory syncytial virus (HRSV) is the most important viral agent of pediatric respiratory tract disease worldwide and also is important in adults in general and in the elderly and hematopoietic stem cell transplant recipients in particular. There is no approved vaccine or effective antiviral therapy. We previously developed a method for producing infectious RSV entirely from cloned cDNAs (?reverse genetics?), whereby defined changes can be introduced into infectious virus via the cDNA intermediate. We have used this technique extensively to characterize the molecular biology and pathogenesis of the virus and to develop live attenuated viruses for use as a live intranasal pediatric vaccine. Vaccine development for HRSV is difficult because immunization should begin by 1 to 2 months of age, a time in life when immune responses are reduced due to immunologic immaturity and the immunosuppressive effects of maternally-derived serum antibodies. [unreadable] As described in an accompanying report (B. R. Murphy et al), we developed several recombinant vaccine candidates that have been evaluated in clinical trials. One virus, called rA2cp248/404/1030/delSH, was safe and immunogenic in young infants. However, approximately one-third of the vaccine virus isolates recovered from vaccinees had evidence of a partial loss of the temperature-sensitive (ts) phenotype. Sequence analysis indicated that this was associated with reversion and loss of either the 248 or the 1030 point mutation. Reversion can readily occur because each amino acid substitution is based on a single nucleotide substitution relative to wild type. Amino acid substitutions based on two or three nucleotide changes differences relative to wild type would be correspondingly less susceptible to reversion. We presently have constructed panels of viruses in which either the 248 or the 1030 locus has been modified so as to represent each of the 20 possible amino acid assignments, resulting in 20 viruses per locus. We will now analyze each of these to determine the ts phenotype in vitro and the attenuation phenotype in mice. With this information, and taking advantage of the degeneracy of the genetic code, we will choose a codon for each locus that encodes an attenuating amino acid assignment and differs by as many nucleotides as possible from codons for non-attenuating assignments. This should enable us to construct an improved version of rA2cp248/404/1030/delSH that should have increased genetic and phenotypic stability.[unreadable] When preparing vaccine material for clinical trials, it is increasingly essential to minimize the use of animal products (to preclude adventitious agents such as the bovine spongiform encephalopathy agent), to avoid introduction of heterologous virus, and to use a cell line that is approved and validated for human use. We developed the capability to produce recombinant, attenuated HRSV completely from plasmids in approved Vero cells, eliminating the need to use vaccinia helper virus. Extensive experimentation identified conditions that produce recombinant virus with high efficiency. This removes a hurdle to developing vaccine candidates in our laboratory.[unreadable] The HRSV G protein is a heavily glycosylated species that is expressed both as a transmembrane surface protein and as a form that lacks the membrane anchor and is secreted. In addition, the G ectodomain contains a conserved cysteine noose that contains a sequence motif that appears to be a mimic of the CX3C chemokine called fractalkine. Studies in a number of laboratories indicate that HRSV G is deficient in epitopes for CD8+ cytotoxic T cells (CTLs) in mice and humans. However, we found that recombinant HRSV from which the G gene had been deleted induced a low level of CD8+ CTL in mice compared to wild type HRSV. Similar results were observed with recombinant viruses that had been modified to ablate expression of the secreted from or from which the conserved cystine noose had been deleted. This suggests that the secreted form of G bearing the cystine noose play an important role in generating HRSV-specific CTL. The mechanism by which this effect occurs remains unknown and is under investigation. [unreadable] We previously showed that the HRSV NS1 and NS2 proteins inhibit the human type I interferon (IFN) response. The effect of these proteins on viral attenuation and the host immune response is of particular interest because viruses lacking these genes are under clinical evaluation as promising vaccine candidates. We have now shown that this inhibition extends to the murine system, validating the mouse model for studying the immunobiology of these mutants. The one difference is that, in the human system, NS1 plays the greater role in inhibiting the interferon response whereas in mice it is NS2. Viruses lacking NS1 or NS2 should induce a more robust interferon response, and since some of the genes that are up-regulated by type I interferon encode proteins that are involved in innate and adaptive immunity, this might have qualitative or quantitative effects on the immune response. Consistent with this supposition, we found that virus lacking the NS2 protein induced a more robust CD8+ CTL response in BALB/c mice, whether analyzed by tetramer staining, IFN gamma production, or in vitro cell killing. This effect was not observed in STAT1 knock out mice, consistent with the idea that the host interferon system is involved in the increase. This suggests that mutants lacking NS1 (which in the human system appears to play the role that NS2 does in the mouse) may be associated with increased stimulation of CD8+ CTLs in humans. This might be important because, in the mouse model, interferon gamma expressed by CD8+ CTL is important in regulating the CD4+ T cell response and is associated with reduced vaccine reactogenicity. [unreadable] Methods: Cell culture; virus propagation; virus titration; DNA transfection; cDNA construction and cloning; site-directed mutagenesis; synthetic oligonucleotides, peptides, and genes; automated and manual DNA sequence analysis; recombinant protein expression; Northern blots; Western blots; immunoprecipitation; immunofluorescence; cytokine ELISA assays; studies of virus replication, immunogenicity, pathogenesis and protective efficacy in rodents ; serological analysis including ELISA and plaque reduction assays; PCR; real time RT-PCR; confocal microscopy; luciferase. [unreadable] Goals and Objectives: The primary goal of this project is to develop a live attenuated vaccine for human respiratory syncytial virus (HRSV) designed for intranasal administration to infants and young children. A second goal is to understand the basis of attenuation for candidate vaccines, and to exploit the study of viral mutants in pre-clinical and clinical settings to learn more about viral biology and the host response. [unreadable]